Protein kinases are enzymes that serve as key components of signal transduction pathways by catalyzing the transfer of the terminal phosphate from ATP to the hydroxy group of tyrosine, serine and threonine residues of proteins. As a consequence, protein kinase inhibitors and substrates are valuable tools for assessing the physiological consequences of protein kinase activation. The overexpression or inappropriate expression of normal or mutant protein kinases in mammals has been demonstrated to play significant roles in the development of many diseases, including cancer, inflammation and diabetes.
Protein kinases can be divided into two classes: those, which preferentially phosphorylate tyrosine residues (protein tyrosine kinases) and those, which preferentially phosphorylate serine and/or threonine residues (protein serine/threonine kinases). Protein tyrosine kinases perform diverse functions ranging from stimulation of cell growth and differentiation to arrest of cell proliferation. They can be classified as either receptor protein tyrosine kinases or intracellular protein tyrosine kinases. The receptor protein tyrosine kinases, which possess an extracellular ligand binding domain and an intracellular catalytic domain with intrinsic tyrosine kinase activity, are distributed among 20 subfamilies.
Receptor tyrosine kinases of the epidermal growth factor (“EGF”) family, which includes HER-1, HER-2/neu and HER-3 receptors, contain an extracellular binding domain, a transmembrane domain and an intracellular cytoplasmic catalytic domain. Receptor binding leads to the initiation of multiple intracellular tyrosine kinase dependent phosphorylation-processes, which ultimately results in oncogene transcription. Breast, colorectal and prostate cancers have been linked to this family of receptors.
FLT-3 (FMS-like tyrosine kinase 3) is a member of the receptor tyrosine kinase family that includes PDGFR, c-kit, and c-fms. FLT-3 kinase is expressed mainly by early myeloid and lymphoid progenitor cells and is involved in the proliferation, differentiation, and apoptosis of hematopoietic cells. FLT-3 kinase is activated by FLT-3 ligand, which upon binding to the receptor causes dimerization and autophosphorylation and subsequent activation of the kinase domain, which subsequently then activates the phosphoinositol-3-kinase (PI3K), STAT and RAS signal transduction pathways.
Platelet derived growth factor (“PDGF”) receptors mediate cellular responses that include proliferation, migration and survival and include PDGFR, the stem cell factor receptor (c-kit) and c-fms. These receptors have been linked to diseases such as atherosclerosis, fibrosis and proliferative vitreoretinopathy.
Insulin receptor (“IR”) and insulin-like growth factor I receptor (“IGF-R”) are structurally and functionally related but exert distinct biological effects. IGF-R expression has been associated with breast cancer.
c-Met serves as the high affinity receptor for hepatocyte growth factor (HGF), signalling through which leads to proliferation, scattering and branching morphogenesis. Over-expression of c-Met has been linked to a number of cancers including hereditary papillary renal carcinomas, ovarian cancer, and head and neck squamous cell carcinomas.
Fibroblast growth factor (“FGR”) receptors consist of four receptors that are responsible for the production of blood vessels, for limb outgrowth, and for the growth and differentiation of numerous cell types.
Vascular endothelial growth factor (“VEGF”), a potent mitogen of endothelial cells, is produced in elevated amounts by many tumors, including ovarian carcinomas. The known receptors for VEGF, flt and KDR, are designated as VEGFR-1 (Flt-1), VEGFR-2 (KDR), VEGFR-3 (Flt-4). A related group of receptors, tie-1 and tie-2 kinases, have been identified in vascular endothelium and hematopoietic cells. VEGF receptors have been linked to vasculogenesis and angiogenesis.
Intracellular protein tyrosine kinases are also known as non-receptor protein tyrosine kinases. Over 24 such kinases have been identified and have been classified into 11 subfamilies. The serine/threonine protein kinases, like the cellular protein tyrosine kinases, are predominantly intracellular.
Cyclin-dependent kinases (“CDK”) are serine/threonine kinases, which are key regulatory components cell cycle entry and progression. Each CDK is associated with a regulatory subunit (cyclin) and together each complex mediates a particular stage of the cell cycle. Inhibitors of CDKs have potential uses in the treatment of cancer.
Mitogen-activated protein (“MAP”) kinases are serine/threonine kinases that can be divided into three groups in mammalian cells: extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAP kinase. p38 MAP kinases play key roles in cellular responses to external stress signals, while inhibitors have anti-inflammatory roles in key animal models of inflammation. The ERK pathway is an important determinant in the control of cell growth, cell differentiation and cell survival. This pathway is up-regulated in human tumors and represents a target for anticancer therapy.
Aurora kinases are a family of serine/threonine kinases that are important regulators of the mitotic cellular division process (Bischoff J R, Plowman G D: Trends Cell Biol. 1999 November; 9(11): 454-9 and Carmena M, Eamnshaw W C: Nat Rev Mol Cell Biol. 2003 November; 4(11): 842-54). Expression of all Aurora kinases peaks during the G2 and M phases of the cell cycle, while being low in cells at rest. Inhibitors of the Aurora family of kinases may block growth of all cancer types (Katayama H4, Brinkley W R, Sen S: Cancer Metastasis Rev. 2003 December; 22(4): 451-64 and Harrington E A, Bebbington D, et al.: Nat Med. 2004 March; 10(3): 262-7).
Members of the Aurora family of kinases [Aurora-1 (“A”), Aurora-2 (“B”) and Aurora-3 (“C”)] are highly homologous, especially within the C-terminal domain, although they differ in the length and sequence in the N-terminal domain (Bischoff J R, Anderson L, et al.: EMBO J. 1998 Jun. 1; 17(11): 3052-65 and Giet R, Prigent C: J Cell Sci. 1999 November; 112 (Pt 21): 3591-601). As the three Aurora kinases have nearly identical ATP binding sites, inhibitors, which bind at this location, may be expected to inhibit all three Aurora subtypes.
Aurora-1 is localized in the centrosome and in the spindle midzone and midbody (Sugimoto K, Urano T, et al.: Cell Struct Funct. 2002 December; 27(6): 457-67). Aurora-1 plays a critical role in mitotic spindle formation and centrosome maturation, which ensures accurate separation of chromosomes into each daughter cell (Giet R, McLean D, et al.: J. Cell Biol. 2002 Feb. 4; 156(3): 437-51). Overexpression of Aurora-1 transforms fibroblasts and yields aneuploid cells that contain multiple centrosomes and multipolar spindles (Giet R, Prigent C: J Cell Sci. 1999 November; 112 (Pt 21): 3591-601). Aurora-1 maps to 20q13.2 in humans, a region that is amplified in some primary tumors (Tanner M M, Grenman S, et al.: Clin Cancer Res. 2000 May; 6(5): 1833-9). Overexpression of the Aurora-1 kinases has been detected in a wide range of tumor types including: colorectal, gastric, ovarian and breast (Sakakura C, Hagiwara A, et al.: Br J Cancer. 2001 Mar. 23; 84(6): 824-31; Takahashi T, Futamura M, et al.: Jpn J Cancer Res. 2000 October; 91(10): 1007-14; Gritsko T M, Coppola D, et al.: Clin Cancer Res. 2003 April; 9(4): 1420-6 and Tanaka T, Kimura M, et al.: Cancer Res. 1999 May 1; 59(9): 2041-4).
Aurora-2 kinase is a protein associated with chromosomes that is important for chromosomal biorientation on the mitotic spindle and in the regulation of kinetochore-microtubule interactions (Ditchfield C, Johnson V L, et al.: J. Cell Biol. 2003 Apr. 28; 161(2): 267-80). Aurora-2 kinase is overexpressed in various tumor cell types and increases in line with the Duke stage of primary colorectal cancer (Katayama H, Ota T, et al.: J Natl Cancer Inst. 1999 Jul. 7; 91(13): 1160-2).
Aurora-3 kinase is collocated to the testis in normal tissue, but has been shown to be overexpressed in a high percentage of colorectal cancers (Takahashi T, Futamura M, et al.: Jpn J Cancer Res. 2000 October; 91(10): 1007-14). Aurora-3 kinase exhibits a centrosomal location from anaphase through telophase.
Small molecule kinase inhibitors of the Aurora kinases have been described, one of which inhibited the proliferation of a wide range of tumor cell types and suppressed tumor growth in vivo (Ditchfield C, Johnson V L, et al.: J. Cell Biol. 2003 Apr. 28; 161(2): 267-80; Harrington E A, Bebbington D, et al.: Nat Med. 2004 March; 10(3): 262-7 and Hauf S, Cole R W, et al.: J. Cell Biol. 2003 Apr. 28; 161(2): 281-94). The cell death was due to apotosis and was associated with an inhibition of Ser10 phosphorylation on Histone H3, which is a downstream target of the Aurora kinases.